The present disclosure relates generally to methods for generating single-stranded DNA molecules of defined sequence and length from template containing a target nucleotide sequence. Specifically, the present disclosure provides a method for generating short single-stranded DNA molecules of defined sequence and length by linear or non-linear amplification of a template using specially designed primers or probes, conversion of double-stranded amplification products into single-stranded amplification products if necessary, and trimming single-stranded amplification products to yield the desired DNA molecule of defined sequence and length.
Amplification of Target Sequences
A number of methods have been developed for amplification of target nucleotide sequences in nucleic acid templates. These include the polymerase chain reaction (PCR), rolling circle amplification (RCA), ligase chain reaction (LCR), self-sustained sequence replication (3SR), nucleic acid sequence based amplification (NASBA), and strand displacement amplification (SDA).
Current methods of PCR amplification involve the use of two primers which hybridize to the regions flanking target nucleotide sequence, such that DNA replication initiated at the primers will replicate the target nucleotide sequence. By separating the replicated strands from the template strand with a denaturation step, another round of replication using the same primers can lead to many-fold amplification of the target nucleotide sequence.
Rolling circle amplification (RCA) is an isothermal amplification method in which a circularizable single-stranded probe is hybridized to a template such as RNA or denatured DNA at regions flanking the target nucleotide sequence, the strand is circularized using primer extension and/or ligation, sequences in the circle are then selectively amplified, and optionally, non-circular products are removed by digestion.
Linear and Nonlinear Amplification of Target Sequences
Amplification of target sequences may be carried out in linear or non-linear mode, for example as described in EP 0971039 to Rabanni et al. Linear amplification of target sequences may be used when a starting mixture contains a large number of copies of a target sequence. Generally, linear amplification utilizes a single initial primer, probe, or other nucleic acid construct to carry out the amplification process.
Non-linear amplification of target sites is often used when the number of copies of a target sequence present in the starting mixture is small. Non-linear amplification results in exponential growth in the number of gene copies present. PCR and RCA, especially RCA in the branching mode, can be used effectively in the non-linear amplification mode. (Lizardi et al., 1998, Nature Genetics 19:225-232)
Generation of Single Stranded DNA
Many amplification methods generate double-stranded amplification products, while many applications require single-stranded DNA molecules containing the target sequence. Double-stranded DNA can be converted to single-stranded DNA by separating the strands or by removing one strand of the duplex. Strands of a duplex can be separated by thermal or chemical methods of disrupting interstrand bonds. Removing one strand allows recovery of the desired strand and elimination of its complement. One strategy for selectively removing one strand of a DNA duplex is to use exonuclease digestion, preferably 5xe2x80x2xe2x86x923xe2x80x2 exonuclease digestion, where one strand is protected from attack by the exonuclease.
For example, U.S. Pat. No. 5,518,900 to Nikiforov et al. describes modifying one of two PCR primers used for amplification by incorporating phosphorothioate nucleotide derivatives in the 5xe2x80x2 end of the modified primer, rendering it resistant to exonuclease digestion. After amplifying target sequences using PCR, the double-stranded amplification product is subjected to exonuclease digestion. The unprotected strand is preferentially digested by a 5xe2x80x2xe2x86x923xe2x80x2 exonuclease, leaving a single-stranded product consisting of the other strand.
In an alternate approach, Shchepinov et al. uses branched PCR primers that are resistant to 5xe2x80x2-exonuclease digestion, with the result that exonuclease digestion of the double-stranded amplification products gave single strands protected from digestion by the exonuclease-resistant branched primers. (Shchepinov et al., 1997, Nuc Acids Res 25:4447-4454) Disadvantages of this method are that branched primers are difficult to synthesize and the resulting PCR products are branched.
Another approach to generating single-stranded DNA uses phosphorylation of the 5xe2x80x2 end of one strand of a double-stranded amplification product to produce a preferred lambda exonuclease substrate. (Higuchi et al., 1989, Nuc Acids Res 25: 5685) This method allows selective degradation of the phosphorylated strand and recovery of the nonphosphorylated strand.
Generation of Short Single-stranded DNA Molecules
Short single-stranded DNA molecules of defined sequence and length are needed for applications such as arrays, where the desirable size range is about 45 nucleotides or less. Although methods for generating single-stranded DNA molecules are known in the art, these methods do not necessarily generate small molecules of 45 nucleotides or less. For example, the methods discussed above for generating single-stranded DNA do not provide short single-stranded DNA molecules of defined sequence and length. U.S. Pat. No. 5,518,900 to Nikiforov et al. teaches methods for generating single-stranded DNA molecules from double-stranded PCR amplification products, but the resulting PCR products are typically longer than 45 nucleotides. The method of Shchepinov et al. produces branched PCR products that are typically longer than 45 nucleotides. (Shchepinov et al., 1997, Nuc Acids Res 25:4447-4454) Likewise, the method of Higuchi et al. yields single-stranded DNA products that are not in the desired size range. (Higuchi et al. 1989, Nuc Acids Res 17: 5865)
Shaw and Mok disclose cleaving single-stranded DNA into fragments by interaction with a specially designed oligodeoxyribonucleotide adaptor and the class-IIN restriction endonuclease, XcmI. (Shaw and Mok, 1993, Gene 133:85-89) After hybridizing to the target DNA and addition of XcmI, template DNA is specifically cleaved to near completion; however, hairpin structures on the template close to the hybridization site reduce the efficacy of cleavage.
The invention described herein is directed to methods for generating a single-stranded DNA molecule of defined sequence and length, where the method includes amplification, conversion, and trimming steps. In accordance with one aspect of the invention, amplification of a template having at least one target nucleotide sequence is directed by one or more primers having at least one exogenous nucleotide sequence not present in the target nucleotide sequence, where the amplification step generates amplification products with at least one target nucleotide sequence and at least one exogenous nucleotide sequence introduced by the primer. In accordance with another aspect of the invention, a conversion step may be performed. When the amplification step generates double-stranded amplification products, the method includes a conversion step wherein each double-stranded amplification product is converted to a single-stranded amplification product. When the amplification step generates single-stranded amplification products, the conversion step is not required. In accordance with another aspect of the invention, the single-stranded amplification product is trimmed to generate a single-stranded DNA molecule of defined sequence and length.
In accordance with one aspect of the invention, polymerase chain reaction (PCR) is used for the amplification step to produce double-stranded amplification products. In one embodiment, multiplex PCR may be used. The amplification step can be carried out in linear or non-linear mode. The template for amplification may be genomic DNA, cDNA, or RNA.
In accordance with another aspect of the invention, rolling circle amplification (RCA) is used for the amplification step. In various embodiments, RCA may produce double-stranded or single-stranded amplification products. In one embodiment, RCA in the linear mode is used to generate single-stranded amplification products. The amplification step can be carried out in linear or non-linear mode. The template for amplification may be genomic DNA, cDNA, or RNA, including mRNA.
In one embodiment, primers for the amplification step may have an addressable ligand such as biotin attached to the primer. In another embodiment, exogenous nucleotide sequence introduced by primers used in the amplification step may contain self-complementary sequences that form hairpin structures. These self-complementary sequences that form hairpin structures may contain at least one restriction enzyme recognition site for a restriction enzyme involved in the trimming step, and suitable restriction enzymes include Type II restriction enzymes such as EcoRI, or Type IIS restriction enzymes such as FokI.
In another embodiment, exogenous nucleotide sequence(s) introduced by primers include sequence(s) that can form a recognition site for a restriction enzyme involved in said trimming step, where the restriction enzyme recognition site is formed upon addition of at least one auxiliary oligonucleotide. Suitable restriction enzymes include Type II restriction enzymes such as EcoRI, or Type IIS restriction enzymes such as FokI. In another embodiment, the auxiliary oligonucleotide includes at least one sequence having an addressable ligand such as biotin attached.
In accordance with another aspect of the invention, the conversion step may be carried out by digesting one strand of a double-stranded amplification product using a 5xe2x80x2xe2x86x923xe2x80x2 exonuclease such T7 or lambda exonuclease, where the amplification product includes at least one target nucleotide sequence and at least one exogenous nucleotide sequence introduced by a primer during the amplification step. In a preferred embodiment, the exogenous nucleotide sequence introduced by a primer includes modified nucleotides that confer resistance to digestion using 5xe2x80x2xe2x86x923xe2x80x2 exonuclease, for example where the nucleotides are phosphorothioate derivates. In another preferred embodiment, the exogenous nucleotide sequence introduced by a primer includes modified nucleotides that confer sensitivity to digestion using 5xe2x80x2xe2x86x923xe2x80x2 exonuclease, for example where the modified nucleotides are phosphorylated.
In accordance with another aspect of the invention, a method is provided for generating a single-stranded DNA molecule of defined sequence and length which avoids the exonuclease step and a requirement for auxiliary oligonucleotides. The method includes amplifying a template containing at least one target nucleotide sequence, where the amplification is directed by at least one primer having at least one exogenous nucleotide sequence not present in the target nucleotide sequence, generating a plurality of double-stranded amplification products having at least one target nucleotide sequence and at least one exogenous nucleotide sequence introduced by at least one primer, then nicking each double stranded amplification product at one end of a defined sequence and cleaving the double stranded amplification product at the other end of a defined sequence to generate a DNA molecule of defined sequence and length, and finally, separating the single stranded DNA molecule of defined sequence and length from the remainder of the amplification product that includes its complement and the primer duplexes of the amplification product. The single stranded DNA molecule of defined sequence and length can be recovered for further use. In accordance with one aspect, the single stranded DNA molecule of defined sequence and length is separated from the remainder of the amplification product by heating under conditions that allow the single stranded DNA molecule of defined sequence and length to separate from its complement while leaving the the primer duplexes of the amplification product intact. In accordance with another aspect, the primers include an addressable ligand attached to the primer. In one embodiment, the adressable ligand is biotin, and the remainder of the amplification product can be removed by attachment to magnetic beads carrying streptavidin that binds to biotin labels attached to the 5xe2x80x2 end of at least one primer.
In accordance with the methods of the present invention, the single-stranded DNA molecule of defined sequence and length generated by the present invention may be between 10 and 100 nucleotides, or between 10 and 50 nucleotides in length. In one embodiment, the single-stranded DNA molecule of defined sequence and length is 15 nucleotides in length. In another embodiment, the single-stranded DNA molecule of defined sequence and length is 17 nucleotides in length. In yet another embodiment, the single-stranded DNA molecule of defined sequence and length is 21 nucleotides in length. In yet another embodiment, the single-stranded DNA molecule of defined sequence and length is 30 nucleotides in length.
Another aspect of the present invention is directed to methods for identifying an organism or individual using some or all of the following steps: 1) obtaining template having at least one target nucleotide sequence; 2) amplifying the template in an amplification reaction directed by at least one primer having an exogenous nucleotide sequence not present in the target nucleotide sequence; 3) generating amplification products having at least one target nucleotide sequence and at least one exogenous nucleotide sequence introduced by a primer; 4) converting double-stranded amplification products to single-stranded amplification products; trimming each single-stranded amplification product to generate a single-stranded DNA molecule of defined sequence and length; 5) determining the mass or nucleotide sequence of each single-stranded DNA molecule of defined sequence and length; and 6) using at least one mass or nucleotide sequence determination of at least one single-stranded DNA molecule of defined sequence and length to identify at least one organism or individual. In accordance with another aspect of the invention, it is understood that if the amplification step produces single-stranded amplification products, the conversion step is not required. In one embodiment, mass spectroscopy may be used to determine the mass or nucleotide sequence of each single-stranded DNA molecule of defined sequence and length. In another embodiment, a multiplicity of individuals or organisms is identified by this method.